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Sheep anti Green Fluorescent Protein antibody used for the identification of GFP tagged VEGF-A expressing cells by immunofluorescence.
The HSV-1 transactivator ICP4 is required for VEGF-A expression.
Human 293 cells were plated and infected with 3 pfu per cell HSV-1 strain McKrae. Fold induction of VEGF-A transcript expression was determined via geometric means of fold induction of VEGF-A transcript relative to the housekeeping genes β-actin, TBP and PPIA. (A) Inhibition of protein synthesis with cycloheximide (100 μg/mL) blocked upregulation of VEGF-A following HSV-1 infection while (B) inhibition of viral DNA synthesis with acyclovir (200 μM) had no significant effect. (C) ICP4 dependence was tested using human 293 cells infected with 3 pfu per cell of wild type HSV-1 KOS or KOS-derived mutants lacking ICP4 (ICP4- null virus) or the HSV-1 origin binding protein (OBP- null virus). VEGF-A transcript was measured via real time RT-PCR relative to β-actin, TBP and PPIA. (*p<0.05 relative to uninfected control cells). (D) Human 293 cells transfected with pVA8855 or the promoterless luciferase control plasmid, pGL3. Luciferase activity was measured at 12 hours PI with HSV-1 KOS, ICP4- null, or OBP- null virus. (E) VEGF-A mRNA levels in human 293 cells infected with 3 pfu per cell HSV-1KOS or an HSV-1 ICP0- null virus that fails to make the IE co-transactivator protein, ICP0; both viruses significantly up-regulated VEGF-A mRNA levels. (F) Mice lacking type I interferon responses due to deficiency in the type I interferon receptor (CD118-/-) were infected with HSV-1 KOS or HSV-1 ICP0- null virus. Corneas were harvested at 24 hours PI with 105 pfu of HSV-1 and analyzed for VEGF-A levels by cytokine bead array, expressed as pg of VEGF-A per mg of cornea wet mass. VEGF-A was induced by inoculation with HSV-1 KOS or HSV-1 ICP0- null virus (** p<0.01 relative to uninfected, scarified controls). (G) Reporter mice expressing GFP under the human VEGF-A promoter were analyzed for HSV-1 antigen (red) and GFP (green) along with DAPI (blue) at 12 hours PI with either HSV-1 KOS or HSV-1 ICP4- virus. A and B are representative figures of two experiments, n?=?3/group/experiment. Fold induction values were normalized to VEGF-A levels in uninfected, vehicle-treated controls. Panels C and D are representative of two experiments, n?=?3/group/experiment. Panel E is a summary of two experiments, n?=?6/group. Panel F is representative of two experiments with an n?=?3/group/experiment. Bars denote mean ± SEM. (** p<0.01, *<0.05, NSD?=?non-significant difference).
From: Wuest T, Zheng M, Efstathiou S, Halford WP, Carr DJJ (2011)
The Herpes Simplex Virus-1 Transactivator Infected Cell Protein-4 Drives VEGF-A Dependent Neovascularization.
PLoS Pathog 7(10): e1002278.